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The second step in the biosynthesis of retinoic acid from its precursor retinol is the irreversible oxidation of retinal. Two main families of enzymes are involved in this reaction - the aldehyde dehydrogenases, and cytochrome P450.

1 .2.2. 1 The Cytochrome P450 Enzymes

Of the multiple forms of cytochrome P450 identified to date, only two are able to efficiently oxidise retinal to retinoic acid - P450 I A 1 and P450 1 A2 (Raner et al., 1996; Roberts et al., 1 992), while all other known forms have negligible retinal-oxidising activity (Raner et al., 1 996). The active forms can utilise all-trans, 9-cis, and 1 3-cis retinal; however, P450 l Al is by far the more active, and the expression of the P450 I Al gene has been demonstrated in mouse embryos as early as day 7 of gestation (Kimura et al., 1 987). A more likely role in vivo for the cytochromes P450, however, lies in the metabolism of retinoids to more polar metabolites, as many P450 isoenzymes have the ability to derivatise the carbon at position 4 in the ionone ring (Duester, 1 996). The ability of the P450 enzymes to convert retinoic acid to such metabolites as retinoyl �-glucuronide, 5,6-epoxyretinoic acid, 4-hydroxyretinoic acid, 4-oxoretinoic acid and

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3,4-didehydroretinoic acid has been well established (Eckhoff et ai. , 1 99 1 ; Heyman et aI. , 1992; Levin et ai. , 1 992b; McCormick et ai. , 1 978; Skare et ai. , 1 982; Tang & Russell, 1 990; Thaller & Eichele, 1990). Some of these metabolites may be active in retinoic acid function (e.g. 3, 4-didehydroretinoic acid, 4-oxoretinoic acid), whereas many are likely to be catabolic products destined for excretion. A likely role for the cytochrome P450 enzymes is in the direct metabolism of retinoic acid. Retinoic acid metabolism by P450 enzymes has also been demonstrated to occur when retinoic acid is bound to CRABP (Fiorella & Napoli, 1 991), though it still is not certain what the precise role of CRABP is in this process (see section 1 .3 .3).

1 .2.2.2 The Aldehyde Dehydrogenase Enzymes

In general, members of the aldehyde dehydrogenase (AlDH) family exhibit a broad substrate specificity, and can oxidise various biogenic and xenobiotic aliphatic and aromatic aldehydes (Ambroziak & Pietruszko, 1 99 1 ; Goedde & Agarwal, 1 990; Hsu & Chang, 1 99 1 ; Sladek et aI., 1989; Yoshida et ai. , 1991). Only a limited number of identified AlDH enzymes are able to oxidise retinal - human and sheep AlDH 1 (Klyosov et ai. , 1996; Yoshida et ai. , 1992; this thesis), mouse AHD-2 (Dockham et ai., 1992; Lee et aI., 1 99 1 ), and a number of novel enzymes isolated from rat and mouse tissues. These include rat kidney RALDH-l (Bhat et ai. , 1 995; Labrecque et al. , 1 993; Labrecque et aI. , 1997; Labrecque et ai. , 1 995), mouse RALDH-2 (Zhao et aI., _{1 996),}

rat liver RalDHl (el Akawi & Napoli, 1 994; Posch et ai. , 1992), and rat testis RalDH2 (Wang et ai. , 1 996).

A number of observations link class 1 AlDH to retinoic acid production in vertebrate animals. High endogenous levels of retinoic acid are found in the retina of the embryonic mouse, an area in which AlDH 1 activity is also localised (McCaffery et ai. , 1 992). In zebrafish embryos, AlDH 1 activity co-localises with retinoic acid levels (Marsh­ Armstrong et aI., 1 994), and AlDH 1 was shown to be expressed in the basal forebrain, early in the development of mouse embryos (McCaffery & Drager, 1 994). Early work showed that enzyme-catalysed retinal oxidation is restricted to the cytosolic fraction of mouse liver (Lee et ai. , 1991). This activity is 90 % NAD+ dependent, and 90 % of the NAD+ dependent activity was identified as AHD-2 - the mouse equivalent of human AlDH 1 . The remaining portion of the NAD+ dependent activity was shown to be due to AHD-7 and xanthine oxidase (dehydrogenase form). The Km values of these three enzymes for retinal were determined to be 0.7 fJ.M (AHD-2), 0.6 fJ.M (AHD-7), and 0.9 fJ.M (xanthine oxidase). The non-NAD+ dependent activity is likely to be due to

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aldehyde oxidase, as it is largely inhibited by the aldehyde oxidase inhibitor pyridoxal. However, the major retinal-oxidising activity of mouse liver was identified to be a class 1 cytosolic dehydrogenase. These experiments agreed with similar work using rat liver (Leo et al. , 1 989), rat kidney (Napoli & Race, 1 987), and human keratinocytes (Siegenthaler, 1990). In an experiment using human liver cytosol, in contrast to the majority of studies which use murine tissues and subcellular fractions, all aldehyde dehydrogenase activities were chromatographically isolated, using various aldehydes as substrates for activity (Dockham et al., 1992). From the seven distinct aldehyde

dehydrogenase activities separated, only one could oxidise retinal - AIDH 1 .

Although the studies mentioned above all identify AlDH 1 as playing a major role in retinal oxidation, a number of papers, while identifYing AlDH 1 activity as important, found other enzyme activities to play a significant role (Bhat et al., 1 988b; Chen et al. ,

1995; Kishore & BoutweU, 1 980; Leo et al., 1 989; Napoli & Race, 1988). It is likely that these contrasting observations stem from differences in experimental approach; species specific differences, and tissue and cell specific differences. For example, the studies of (Chen et al., 1995) focused on rat conceptal homogenates. Homogenates

from days 10.5, 1 l .5 and 12.5 of gestation each exhibited enzyme activity for retinoic acid production. The production of retinoic acid was reduced by 40-45 %, both by omission ofNAD+, and replacement ofNAni- by NADH or NADPH. In addition, NAD� was a more efficient cofactor than NADP+ Retinoic acid biosynthesis from retinol was inhibited by the alcohol and aldehyde dehydrogenase inhibitor citral, but not by high concentrations of azide, 4-methylpyrazole (ADH 1 inhibitor), or metapyrone (cytochrome P450 inhibitor). The biosynthesis of retinoic acid from retinal was inhibited by citral, but not metapyrone. From these observations, a two step pathway for retinoic acid synthesis in rat embryos was proposed, whereby the conversion of retinol to retinal is catalysed by an NAD�INADP+ dependent retinol dehydrogenase and the conversion of retinal to retinoic acid is catalysed by an NAD+/NADPT dependent retinal dehydrogenase and a retinal oxidase.

There is a strong case for cytosolic aldehyde dehydrogenase being the major enzyme involved in retinal oxidation in the human. It was previously thought that the principle role of AlDH 1 was the oxidation of acetaldehyde derived from the oxidation of ingested ethanol (Greenfield & Pietruszko, 1 977). However, the reported Km of AIDH 1 for acetaldehyde varies from 22 to 483 !J,M (Rashkovetsky et ai. , 1994), while typical

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1 996). Reported Km values for acetaldehyde oxidation by another major AIDH found in

liver, mitochondrial AIDH 2, range from <0. 1 I-lM to 9 I-lM, making it highly likely that

this isozyme has acetaldehyde oxidation as its main function (Rashkovetsky et al., 1 994).

The ability of AlDH 1 to oxidise retinal has been well documented (Dockham et al.,

1 992; Klyosov et al., 1 996; Rashkovetsky et al., 1 994; Yoshida et al. , 1 992; Yoshida

et al. , 1 993). In addition, AlDH 1 is the only identified liver enzyme with retinal­

oxidising ability in humans, as AlDH 2 is unable to accept retinal as a substrate (Y oshida

et al., 1 992). The reported Km values of AIDH 1 for all-trans retinal range from

0.06 I-lM (Y oshida et ai. , 1 993) to 1 . 1 I-lM (KJyosov et ai. , 1 9 96), and the catalytic

efficiency for retinal is 1 00-500 times greater than for acetaldehyde (KJyosov et al. ,

1 996; Yoshida e t al., 1 993). This means that, with an estimated biological concentration

of retinal of -0. 1 I-lM, retinal is an excellent candidate for the primary physiological

substrate of AlDH 1 .

1.2.2.3 The Retinal Dehydrogenase Enzymes

Recently, four retinal-specific dehydrogenases from rat and mouse tissues have been

identified - RalDHl (el Akawi & Napoli, 1 994; Posch et al., 1 992), RalDH2 (Wang et

al., 1 996), RALDH-1 (Bhat et al., 1 995; Labrecque et al., 1 993; Labrecque et al. ,

1 995), and RALDH-2 (Zhao et al. , 1 996). The isolation of RalDHl from rat liver

cytosol was the first reported retinal-specific aldehyde dehydrogenase (Posch et al.,

1 992). RalDHl is tetrameric (214 kDa native MW), with a monomer size of 5 5 kDa

estimated by SDS-PAGE, and a basic isoelectric point (8.3). NAD� dependent RalDHl

accepts as substrates free all-trans retinal and 9-cis retinal, aB-trans retinal bound to

CRBP, and retinal generated from retinol by microsomes, but does not utilise 1 3 -cis

retinal (el Akawi & Napoli, 1 994; Posch et al., 1 992). Allosteric kinetics were observed

for all substrates, and substrate inhibition was seen at retinal concentrations greater then

6 I-lM (Posch et al. , 1 992). A chromatographically similar enzyme activity to RalDHl

was observed in both rat kidney cytosol, and rat testis cytosol (Posch et al. , 1 992). A further novel retinal-oxidising enzyme (RALDH-l ) was isolated from the cytosol of

rat kidney (Labrecque et al., 1 993). RALDH-l has a native molecular weight of

1 40 kDa as determined by size exclusion chromatography, a sub unit molecular weight of

53 kDa by SDS-PAGE, and a basic isoelectric point of 8 . 5 (Labrecque et al., 1 993).

Aldehyde dehydrogenases are typically dimeric (class 3) or tetrameric (classes 1 and 2);

however, the quaternary structure of RALDH-l cannot be estimated from these data. The method used to estimate the native molecular weight has its limitations; in particular,

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the effective Stokes radius of reference and test proteins has a great bearing on how they run through gel filtration columns. In this case no mention of any salt in the gel filtration buffers was made, the absence of which may increase random hydrophobic and ionic interactions by some proteins with the column matrix. It is likely therefore that this enzyme is in fact, tetrameric. RALDH- l utilises acetaldehyde, free aU-trans retinal, 9-cis

retinal, and l l -cis retinal, but not 1 3 -cis retinal as substrates (Labrecque et a!. , 1 993 ;

Labrecque et al., 1 995). Michaelis-Menten kinetics were observed for all substrates, and

substrate inhibition was seen at retinal concentrations greater than 20 !lM. The activity of this enzyme with CRBP-retinal has not been tested, the authors stating that CRBP

does not bind retinal . Although this was originally thought to be the case (MacDonald &

Ong, 1 987; Ong & Chytil, 1 975), it is now accepted that CRBP does bind all-trans

retinal (see section 1 .3 . 1 . 1) . The gene encoding RALDH- l has been cloned from a rat

kidney cDNA library (Bhat et al., 1 995). The full length isolated cDNA contains a

23 1 5 bp open reading frame, encoding a deduced protein of 5 0 1 amino acids. Northern blotting experiments identified this gene to be expressed largely in kidney, followed by lungs, testis, intestine, stomach and trachea, with only a small amount in liver. The absence of retinal-oxidising activity and constitutive AlDH activity from rat liver had

been noted previously (Bhat et al. , 1 995; B hat et al. , 1 988b; Lindahl & Evces, 1 984;

Tottmar et al., 1 973).

Attempts to clone RalDH 1 from a rat liver library were initially confounded by the presence of more abundant AlDH enzymes such as the rat phenobarbital-inducible

enzyme (Wang et al., 1 996). Recently, the cDNA encoding this enzyme has been cloned

using oligonucleotides designed to peptides purified during the initial purification

(Penzes et al., 1 997). The expressed and purified protein showed similar characteristics

to the protein originally purified from rat liver (see Table 1 .2) . While screening a rat testis library for RalDHl , a previously unknown aldehyde dehydrogenase was cloned,

which was named RalDH2 (Wang et al. , 1 996). Expressed and purified protein

recognised free all-trans retinal, and retinal bound to CRBP as substrates, but not acetaldehyde, and the preferred cofactor was NAD"'. Michaelis-Menten kinetics were observed for all substrates. Northern blotting experiments showed that RalDH2 was

expressed solely in testis, but RNase protection assays identified small amounts « 1 0 %

of that in testis) in lung, brain, heart, liver and kidney.

A fourth retinal-oxidising enzyme has been isolated (Zhao et al., 1 996), which is thought

1 -2 1 MM RM H I 0 M 3 _Class 3 R 3 H 3 H 7 B 2 H o 2 M2 Class 2 R 2 H 2 HX RAL DH - 2

]

RALDH R a l D H 2 Type 2 BAl S I H I Class 1 and 2 H o l RALDH - l R a l D H l Class 1 M l R I C l C RY H 6 Y C l YM l Class 1 and 2 E l outIiers H 9

32 aldehyde dehydrogenase sequences were aligned using the GCG program 'pileup'. The above diagram is a pictorial representation of the clustering relationships used in the construction of the sequence alignment (see Chapter 5). The diagram represents similarity between primary amino acid sequences. The retinal oxidising enzymes are - the retinal specific enzymes RALDH- l and -2, RaIDHI and 2, and the class 1 enzymes H I , S I , R I ,

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Table 1.2: A

of the Characteristics of Six

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